Gayana Bot. 68(2),68(2): 2011188-195, 2011 ISSN 0016-5301

Aluminium tolerance in the tropical leguminous N2-fixing shrub angustissima (Mill.) Britton & Rose inoculated with Sinorhizobium mexicanum

Tolerancia al aluminio en la leguminosa arbustiva tropical fijadora de N2 Acaciella angustissima (Mill.) Britton & Rose inoculada con Sinorhizobium mexicanum

REINER RINCÓN-ROSALES1, VÍCTOR M. RUÍZ-VALDIVIEZO2, JOAQUÍN A. MONTES-MOLINA1, FEDERICO A. GUTIÉRREZ-MICELI1 & LUC DENDOOVEN2*

1 Departamento de Biotecnología Vegetal, Instituto Tecnológico de Tuxtla Gutiérrez, Carretera Panamericana km 1080, C.P. 29050.Tuxtla Gutiérrez, Chiapas, México. 2 Laboratorio de Ecología de Suelos, Cinvestav, Av. Instituto Politécnico Nacional 2508, C.P. 07360 México D.F., México. *[email protected]

ABSTRACT

The tropical legume Acaciella angustissima inoculated with or without Sinorhizobium mexicanum ITTG R7T strain was cultivated in an aluminium (Al) spiked soil to study its tolerance towards this metal. Seedlings of A. angustissima were grown in soil with 0, 2, 4 or 6 mg Al kg-1. The effect of Al and inoculation on growth, nodulation, and nitrogen and content in the was monitored. Height and total and dry root weight of plantlets decreased significantly when soil was spiked with Al, but increased when inoculated. Al decreased the number of nodules. Inoculum increased total N and

content of the plantlets. It was found that Al reduced the growth of the tropical leguminous N2-fixing shrub A. angustissima, while inoculation with S. mexicanum ITTG R7T stimulated its growth and increased its tannin and N content.

KEYWORDS: Aluminium tolerance, growth, nodulation, nitrogen and tannins content, Sinorhizobium mexicanum ITTG R7T, soil.

RESUMEN

La leguminosa tropical Acaciella angustissima inoculada con o sin la cepa Sinorhizobium mexicanum ITTG R7T fue cultivada en un suelo tratado con (Al) para estudiar la tolerancia a este metal. Las plantas de A. angustissima fueron crecidas en el suelo con 0, 2, 4 y 6 mg de Al kg-1. Se hizo un seguimiento del efecto de Al e inoculación en el crecimiento, la nodulación, el contenido de nitrogeno y el contenido de taninos en las plantas. La altura, el peso seco total y el peso seco de la raíz de las plantas decrementó significativamente cuando el suelo fue tratado con Al, pero incrementó cuando fue inoculado. El Al disminuyó el número de nódulos. El inóculo incrementó el N total y el contenido de taninos de las plantas.

En este experimento fue encontrado que el Al reduce el crecimiento de la leguminosa arbustiva tropical fijadora de N2 A. angustissima, mientras la inoculación con S. mexicanum ITTG R7T estimula el crecimiento e incrementa el contenido de taninos y nitrógeno.

PALABRAS CLAVE: Tolerancia al aluminio, crecimiento de planta, nodulación, contenido de nitrógeno y taninos, Sinorhizobium mexicanum ITTG R7T, suelo.

INTRODUCTION al. 2004). Below pH 5, Al becomes soluble and is bound 3+ to organic matter or found as Al or Al(OH)3 (Kinraide Intensive agriculture practices, such as the use of large 1991). Aluminium is highly toxic to plants and inhibits their amounts of N fertilizers, acidifies soil (Izaguirre-Mayoral development, decreases biomass production and crop yields + et al. 2002, Poschenrieder et al. 2008). Oxidation of NH4 (Kinraide & Hagerman 2010). Acid soils can be found in derived from urea or ammonium sulphate acidifies the large parts of the world, particularly in the tropics, limiting soil which increases the availability and thus the toxicity food production in most developing countries (Poschenrieder of certain metals, especially aluminium (Al) (Kochian et et al. 2008).

188 Aluminium tolerance of Acaciella angustissima inoculated with Sinorhizobium mexicanum: RINCÓN-ROSALES, R. ET AL.

Al toxicity is associated with changes in the physiological pilot project (http://www.fao.org/news/story/en/item/80060/ and biochemical processes of plants and consequently their icode/). Its goal is to promote the use of plants to productivity (Mora et al. 2006, Meriño-Gergichevich et al. prevent erosion, restore soil fertility and to show how these 2010). The decrease in root growth is one of the initial and provide food, fuel, shelter and income during times of most evident symptoms of Al toxicity in plants (Chen et al. hardship. 2011), thereby reducing the capacity for water and nutrient It remains to be seen, however, if A. angustissima uptake. Above ground plant parts may also be affected by tolerates acid soils and large concentrations of Al. Therefore, Al phytotoxicity (Peixoto et al. 2002, Garzón et al. 2011). seedlings of A. angustissima were inoculated with or At the cellular level, toxic Al triggers an overproduction of without the strain S. mexicanum ITTG R7T and cultivated oxygen reactive species (ROS) in cells (Ma 2005, Meriño- in soil spiked with 0, 2, 4 or 6 mg Al kg-1. Growth, nodule Gergichevich et al. 2010), which alters the functionality formation, total N and tannin content were determined. The of the biomembranes inducing oxidative damage in plants objective of this study was to investigate the tolerance of A. (Boscolo et al. 2003, Cristancho et al. 2011). angustissima inoculated with S. mexicanum towards Al. Although exclusion from root tips and restriction of Al transport to upper plant parts seemed to be the most important mechanisms that allow certain crops and wild MATERIALS AND METHODS plants to grow on acid soils with high Al3+ availability, other species tolerate relatively high Al concentrations not only in BACTERIAL STRAIN roots (Barcelo & Poschenrieder 2002, Panda & Matsumoto Acaciella angustissima plants were inoculated with the S. 2007). mexicanum strain ITTG R7. This strain was isolated recently Phenolic compounds, such as the tannins, flavonols, from nodules of A. angustissima (Lloret et al. 2007). It is flavan-3-ols and anthocyanidins, are characterized by one characterized by a high potential to nodule formation and or more hydroxylated aromatic rings, and they represent a N2-fixation (Rincón-Rosales et al. 2009). The bacteria broad range of plant compounds (Tolrà et al. 2009). The was grown on PY medium (peptone of casein, 5.0 g; yeast concentration of phenolic compounds in plants can increase extract, 3.0 g; CaCl2, 0.6 g; distilled water, 1 l) at 28 ºC and when stressed. For instance, Chen et al. (2011) reported that preserved at 4 ºC until used (Toledo et al. 2003). tea plants increased the amount of phenolic compounds, i.e. catechin, when exposed to elevated Al concentrations. SEED TREATMENT AND GERMINATION Exudation of phenolic compounds by the roots might play Seeds of A. angustissima were scarified with concentrated a role in the exclusion of Al. Phenolic compounds can form H2SO4 for 10 min, surface sterilized with 1 % (v/v) complexes with metals, such as Al, thereby reducing their hypochlorite for 10 min and rinsed eight times with sterile toxic effects (Kochian et al. 2004, Chen et al. 2011). distilled water (Rincón et al. 2003). Treated seeds were Acaciella angustissima (Mill.) Britton & Rose is a germinated on 0.8 % agar-water plates at 28 ºC for 48 h leguminous N2-fixing shrub that was formerly considered (Ruiz-Valdiviezo et al. 2009). as a Mimosa species and later as a member of the Acacia, but has now been classified as belonging to the genera SOIL CHARACTERISTICS, TREATMENT AND EXPERIMENTAL DESIGN Acaciella together with other American (Rico- A clayey soil (type oxisols) was collected at Parral in the Arce & Rodríguez 1998, Rico-Arce & Bachean 2006). A. municipality of Villacorzo, Chiapas, Mexico (16º 11’ N, angustissima can be found in arid and semiarid regions of 93º 16’ W and 580 m altitude). The soil was cultivated with

Mexico (Rzedowski 1978). It is used as firewood, the leaves maize and common bean for > 50 y. The soil with pH H2O as forage for goats and sheep and its bark has traditionally (1:2) 4.6 and EC 0.36 dS m-1, had an organic carbon content been extracted for tannins used to tan hide (Rincón-Rosales of 1.8 g kg-1 soil and a cation exchange capacity of 14.36 & Gutiérrez-Miceli 2008). This legume is characterized by cmole (p+) kg-1 soil. The extractable Al was 3.0 mg kg-1 a tuberous main root with numerous lateral ones, abundant soil. foliage and fast growth (Dzowella 1994, Rico-Arce & Two kg air-dried and 2 mm-sieved soil was sterilized T Bachean 2006). It establishes a symbiosis with N2-fixing to study the specific effects of S. mexicanum ITTG R7 bacteria. Recently, one of the strains that was isolated from strain and added to polyethylene-lined earth pots to avoid A. angustissima was Sinorhizobium mexicanum ITTG R7T contamination. Fifty ml of an aluminium sulfate solution characterized by a large acidity and salinity tolerance (Lloret was added to the soil at four application rates, i.e. 0, 2, 4 or et al. 2007). A. angustissima can grow in nutrient depleted 6 mg Al kg-1 soil. The pots were saturated with deionized saline or acid soils under semi-arid conditions (Ponce- water and stored for 30 days to equilibrate the applied Al. Mendoza et al. 2006). A. angustissima could thus easily Ten seeds scarified as described above were placed in be used to revegetate eroded soil and as such restore soil each pot and thinned to five plants when the seedlings lost the fertility. Concordantly, the FAO has developed the Acacia cotyledon, i.e. approximately 10 d after sowing. Half of the

189 Gayana Bot. 68(2), 2011

seedlings were inoculated with 2 ml of a solution with 1×106 PLANT CHARACTERISTICS cells of S. mexicanum ITTG R7T strain ml-1 while the other Total N in the plants was determined using the Kjeldahl half was left inoculated. A sterile quarter strength Jensen´s method (Bremner 1996). The concentration of Al was medium was added to the plants to provide the basic nutrient determined in a digested 0.5 g plant sub-sample by atomic requirements (Vincent 1970). Each treatment with six plants absorption spectroscopy (Izaguirre-Mayoral 1992). A 30 was replicated six times in a completely randomized design mg sub-sample of each root and shoot was analyzed for in a greenhouse. The soil was irrigated to field capacity with tannin content using the tungsten-molybdenum-phosphorus deionized water throughout the experiment. method (Miranda 2000). Ten days after Al application, two plants were collected at random from each treatment. Damages to the STATISTICAL ANALYSIS different plantlets parts were determined using a photonic Plant characteristics were subjected to one-way analysis of microscope Carl Zeiss Model K-4 (Germany). Plant height variance using PROC GLM (SAS Institute 1989) to test for was measured every 10 days. After 120 days, plants were significant differences between spiking with aluminium and collected and number of nodules counted. The plants were inoculation with S. mexicanum and the least significance oven-dried at 40oC and the dry matter of leaves, stem and difference was then calculated (Table I). The overall effect of roots was measured. The plants were characterized for total the two factors investigated, i.e. aluminium and inoculation, N and tannin content. and their interactions were determined (Table II).

TABLE I. Plant height (cm), total dry plant and root weight (mg), number of nodules and total N and tannin content (mg kg-1) in Acaciella angustissima plants cultivated in a soil spiked with different concentrations of Al and inoculated with or without Sinorhizobium mexicanum.

TABLA I. Altura de planta (cm), peso seco total de planta (mg), peso seco de raíz (mg), número de nódulos, contenido total de N (mg kg-1) y contenido de taninos (mg g-1) en plantas de Acaciella angustissima cultivadas en un suelo adicionado con diferentes concentraciones de Al e inoculado con o sin Sinorhizobium mexicanum. ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ ALUMINIUM CONCENTRATION (mg kg-1 soil) ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ PLANT CHARACTERISTICS INOCULUM 0 2 4 6 LSD a P value ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ Height (cm) No 13.8b 9.5 7.8 6.3 2.1 <0.0001 Yes 24.5 19.8 18.0 15.5 1.8 <0.0001 LSD 1.7 2.8 1.8 2.2 P value <0.0001 <0.0001 <0.0001 <0.0001 Total dry weight (mg) No 0.80 0.62 0.58 0.48 0.14 0.0016 Yes 1.32 1.07 0.97 0.91 0.25 0.0218 LSD 0.16 0.20 0.28 0.26 P value 0.0002 0.0017 0.0143 0.0059 Root dry weight (mg) No 0.43 0.35 0.34 0.24 0.10 0.0134 Yes 0.73 0.63 0.50 0.48 0.14 0.0063 LSD 0.16 0.14 0.15 0.08 P value 0.0043 0.0022 0.0446 0.0004 Number of nodules No NAc NA NA NA NA NA Yes 9.5 4.2 5.7 7.5 2.0 0.0005 LSD NA NA NA NA P value NA NA NA NA Total N (mg kg-1) No 5.7 5.8 6.7 5.5 1.5 0.3756 Yes 14.4 10.9 11.2 11.5 1.0 <0.0001 LSD 0.14 0.09 0.13 0.19 P value <0.0001 <0.0001 0.0002 0.0003 Tannins (mg kg-1) No 0.37 0.46 0.55 0.56 0.09 0.0031 Yes 0.82 0.50 0.58 0.85 0.07 <0.0001 LSD 0.10 0.05 0.09 0.12 P value <0.0001 0.0735 0.4971 0.0010 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ a LSD: Least significant difference (P < 0.05), b Mean of six plants, c ND: Not applicable. / a LSD: Menor diferencia significativa (P < 0.05), b Promedio de seis plantas, c ND: No aplicable.

190 Aluminium tolerance of Acaciella angustissima inoculated with Sinorhizobium mexicanum: RINCÓN-ROSALES, R. ET AL.

TABLE II. Effect of aluminum (ALU) and inoculation with Sinorhizobium mexicanum (INO) and their interactions on plant height (cm), total dry plant and root weight (mg), number of nodules and total N and tannin content (mg g-1) in Acaciella angustissima plants cultivated in the greenhouse.

TABLA II. Efecto del aluminio (ALU) e inoculación con Sinorhizobium mexicanum (INO) y sus interacciones en la altura de planta (cm), el peso seco total de planta (mg), el peso seco de raíz (mg), el número de nódulos, el contenido total de N (mg g-1) y el contenido de taninos (mg g-1) en plantas de Acaciella angustissima cultivadas en invernadero. ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ TOTAL DRY PLANT NUMBER OF FACTOR PLANT HEIGHT WEIGHT ROOT DRY WEIGHT NODULES TOTAL NTANNIN CONTENT ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ ALU 62.53 <0.0001 11.21 <0.0001 11.07 <0.0001 12.62 <0.0001 6.87 0.0017 21.00 <0.0001 INO 517.97 <0.0001 91.46 <0.0001 77.75 <0.0001 NAa NA 425.70 <0.0001 109.26 <0.0001 ALU*INO 0.50 0.6858 0.33 0.8066 1.53 0.2326 NA NA 9.87 0.0002 26.96 <0.0001 ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ a NA: Not applicable/No aplicable.

The relationships between the different plant soil with Al, but the effect was not related to the amount characteristics were visualized by principal component applied. analysis (PCA). Details of the PCA analysis can be found Plant height, total and root dry weight was significantly in Vásquez-Murrieta et al. (2007). Briefly, only principal affected by inoculation and Al added to soil (P < 0.0001) components with Eigenvalues >1 and that explained >10% (Table II). The number of nodules was significantly affected of the total variance were retained. The matrix of 36 columns by the application of Al to soil (P < 0.0001). Inoculation, (6 treatments soils with six plants) and six lines (variables) spiking soil with Al and the interaction between them was used for PCA. All analyses were performed using the significantly affected the concentration of N and tannin in SAS statistical package (SAS 1989). the plantlets (P ≤ 0.0017). The effect of Al on the characteristics of the uninoculated plantlets is clearly shown in scatter plot (Fig. 2). An increased RESULTS Al application rate reduced plant development, i.e. PC1 becomes more negative when the amount of Al added to The A. angustissima plants grown in soil spiked with soil increased, while the tannin content increased, i.e. PC2 Al, but without S. mexicanum, showed visible toxicity increases. PC1 also decreased for the inoculated plantlets symptoms. Microscopic analysis showed that the plants with increased Al content in the soil, but the effect on PC2, had less lateral roots compared to those cultivated in i.e. the tannin content was less clear. untreated soil (Fig. 1). The observed roots were thin with The Al concentration was significantly larger in slightly damaged tips indicating cellular necrosis. The inoculated plants compared to non-inoculated ones and plants treated with S. mexicanum ITTG R7T cultivated increased with increased Al application rates (Table I). in soil spiked with Al showed some damage to the root The Al content in the aerial parts of the non-inoculated A. system, mainly to the lateral roots. The superior part of angustissima plants cultivated in soil spiked with 2, 4 or 6 the roots, however, showed a large number of roothairs mg Al kg-1 soil was significantly 1.7, 1.8 and 3.7 times higher without alterations. than those in cultivated soil with 0 mg Al kg-1, respectively, The height, the total and root dry weight of A. angustissima while in the roots it was 1.6, 1.8 and 4.0 times higher. The decreased with increased application of Al independent of Al content was higher in roots of the inoculated and non- inoculation, while the concentration of tannin increased in inoculated plants compared to that in the aerial parts. The Al the uninoculated plantlets (Table I). The number of nodules, content in the inoculated plants roots was 2.5 times higher total N and the tannin content was also affected by spiking than in the non-inoculated plant roots.

191 Gayana Bot. 68(2), 2011

FIGURE 1. Seedlings of Acaciella angustissima grown in aluminum spiked soil and inoculated with Sinorhizobium mexicanum ITTG R7T. -1 -1 -1 -1 Plant: P1= 0 mg Al kg ; P2= 2 mg Al kg ; P3= 4 mg Al kg and P4= 6 mg Al kg .

FIGURA 1. Plantas de Acaciella angustissima crecidas en un suelo adicionado con aluminio e inoculado con Sinorhizobium mexicanum T -1 -1 -1 -1 ITTG R7 . Planta: P1= 0 mg Al kg ; P2= 2 mg Al kg ; P3= 4 mg Al kg y P4= 6 mg Al kg .

FIGURE 2. Principal component analysis (PCA) performed on characteristics, i.e. tannins content, total N plant, total plant weight, root weight, plant height of Acaciella angustissima spiked with aluminum and inoculated with Sinorhizobium mexicanum ITTG R7T.

FIGURA 2. Análisis de components principales (PCA) realizado sobre las características, i.e. contenido de taninos, N total de planta, peso total de planta, peso de raíz y altura de planta de Acaciella angustissima (Mill.) Britton & Rose adicionado con aluminio e inoculado con Sinorhizobium mexicanum ITTG R7T.

192 Aluminium tolerance of Acaciella angustissima inoculated with Sinorhizobium mexicanum: RINCÓN-ROSALES, R. ET AL.

DISCUSSION the accumulation of Al in the roots of N2-fixing legumes species native grown in the Venezuelan savanna, such Growth of A. angustissima plants was inhibited when as Chamaecrista flexuosa L., Clitoria guianensis Aubl. cultivated in soil spiked with Al and not inoculated with Benth., Galactia jussiaeana Kunth, Zornia curvata Mohl., S. mexicanum, leaf fall was faster and stems were intense Centrosema pubescens Benth., Chamaecrista tetraphylla red-purple and thinner than those of plants cultivated in soil Benth. and Phaseolus gracillis Poepp. ex Benth., did not without Al addition. Similar toxicity symptoms have been inhibit their symbiotic capacity. However, Igual et al. observed in several species of tropical legumes (Hossain et (1997) reported that the N2-fixation efficiency of Casuarina al. 2005, Watanabe et al. 2006, Scott et al. 2008). Silva et cunninghamiana L. plants decreased from 0.20 mg N-fixed al. (2010) reported that the primary symptom of Al toxicity mg-1 nodule dry weight at 0 μM Al to 0.10 mg N-fixed mg-1 in the plants is a rapid, i.e. within minutes, inhibition of root nodule dry weight at 880 μM Al. Nodules on the roots not growth resulting in a reduced and damaged root system, only fix N2 but also help to detoxify the plant (Kochian et al. limited water and mineral nutrient uptake and reduced 2005). Izaguirre-Mayoral et al. (2002) reported the exclusion growth. of Al from nodule tissues of Dioclea guianensis Benth., an The amount of root hairs was lower for plants cultivated Al accumulator legume species native of Venezuelan tropical in Al-contaminated soil. The root hairs are important forests. because they excrete chelating substances that prevent Al3+ Inoculation with S. mexicanum ITTG R7T increased the ions from entering the plant. Root tips of soybean (Glycine total N and tannin content in A. angustissima compared max L.) release citrate, while wheat (Triticum aestivum to plants that were not inoculated. Leguminous species L.) malate (Kinraide & Hagerman 2010, Xu et al. 2010). growing in soil with large amounts of Al increase the Flavonoids such as catechin that protect against toxicity accumulation of tannins in the bark, root and also in of the Al3+ (Nguyen et al. 2003). The citrate, malate and nodules as a protection mechanism (Kochian et al. 2005). flavonoid are organic compounds that form a stable complex Small granules of tannins were even found in the nodules with Al that are not phytotoxic for the plant (de Andrade et suggesting that they might protect the nodules against Al al. 2011). Root hairs also facilitate the communication and toxicity (Barceló & Poschenrieder 2002, Izaguirre-Mayoral molecular recognition between the plant and the rhizobia et al. 2002). Tannins bind to Al3+ ions and exclude it in bacteria (Räsänen et al. 2001) so that nodules can be formed that way (Barceló & Poschenrieder 2002). This protection (Pueppke & Broughton 1999). It has been reported that mechanism is also observed in Acacia auriculiformis A. nodule tissues participate in the exclusion of aluminium Cunn. ex Benth. and Eucalyptus camaldulensis Dehnh. and as such protecting the plant against toxic effects of Al (Nguyen et al. 2003). Rincón-Rosales & Gutiérrez-Miceli (Barcelo & Poschenrieder 2002). (2008) observed that A. angustissima shrubs grown in acid Nodules were formed on the roots of A. angustissima soils with Al-concentration >3 mg Al kg-1 accumulate up to inoculated with Sinorhizobium mexicanum ITTG R7T strain 25% more tannins that those growing in moderate alkaline cultivated in soil spiked with Al. Shirokikh & Shirokikh soils with Al a concentration >1.2 mg Al kg-1. (2007) reported that a Rhizobium leguminosarum bv. Trifolii The accumulation of Al in the roots of A. angustissima, 9-4A strain isolated from red clover (Trifolium pratense mainly in the secondary ones, classified this species as a

L.) can fix N2 in spite of the presence of Al. The presence root-Al accumulator. The legumes Chamaecrista flexuosa of nodules that fix nitrogen positively affects growth of L., Clitoria guianensis Aubl., Galactia jussiaeane Kunth plants (Räsänen et al. 2001). Nodule formation allowed A. and Zornia curvata Mohl. are also considered root-Al angustissima to grow taller than plants without nodules as N2 accumulators (Izaguirre-Mayoral et al. 2002). They grow in was fixed and made the plants more resistant to Al toxicity. soils of the Venezuelan savanna with pH 4 and Al content of Al toxicity normally reduces plant growth. Phytotoxic 8.4 cmol kg-1 and accumulate Al mainly in their roots. studies on the effect of Al on growth of the legume Vigna It was found that the tropical leguminous N2-fixing shrub radiate L. showed that increased Al concentration reduced A. angustissima was tolerant to large amounts of Al in soil so dry weight of the seedlings significantly (Neogy et al. 2002). it could be used to restore soil fertility while inoculation with Du et al. (2009) reported that the growth of the seedlings S. mexicanum ITTG R7T will further increase that resistance of stylo (Stylosanthes spp.) was inhibited when the Al3+ and improve its growth. Nodules not only stimulated growth concentration increased from 50 μM to 100 μM. and plant-N content, but also stimulated tannin production All nodules formed on A. angustissima showed a red protecting A. angustissima against Al toxicity. coloration and reduced acetylene so they fixed N2 (Räsänen et al. 2001). Additionally, the ultra-structural analysis of nodules showed the bacteroid with eight rod-shaped ACKNOWLEDGEMENTS bacteroids indicating that the nitrogenase activity was not inhibited. Izaguirre-Mayoral et al. (2002) reported that The work was done in the laboratory of plant biotechnology

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of Instituto Tecnológico de Tuxtla Gutiérrez (Chiapas, IZAGUIRRE-MAYORAL, M.L., S. FLORES & T. OROPEZA. 2002.

Mexico) while soil and plants were analyzed at the Aluminium tolerance in nodulated N2 Fixing legumes species Instituto Nacional de Investigaciones Forestales, Agrícolas native to two contrasting savanna sites. Plant and Soil 245: y Pecuarias (INIFAP) in Guanajuato (Mexico). We 163-168. KINRAIDE, T.B. 1991. Identity of the rhizotoxic aluminum species. thank A. Chavez and P. Espinosa for field work at the Plant and Soil 134:167-178. Parral (Chiapas), and A. Morales for providing the strain KINRAIDE, T. & A. HAGERMAN. 2010. Interactive intoxicating and T Sinorhizobium mexicanum ITTG R7 . This project was ameliorating effects of tannic acid, aluminum (Al3+), copper funded by Coordinación Sectorial de Desarrollo Académico 2+ 2- (Cu ), and selenate (SeO4 ) in wheat roots. A descriptive and (COSNET) grant No. 867.01P. mathematical assessment. Physiologia Plantarum 139: 68-79. KOCHIAN, L.V., O.A. HOEKENGA & M.A. PIÑEROS. 2004. How Do Crop Plants Tolerate Acid Soils? 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